Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe reversal of immune tolerance represents one central goal in cancer immune therapies and serves as a rationale for developing [3,5]. Furthermore, these drugs can enhance the immunogenicity of the tumor epithelium, and as well change the immunosuppressive cytokine milieu produced by the tumor and its microenvironment, thereby facilitating the maturation and function of effector cells in innate and adaptive immunity [6]. The immunomodulatory effects of established anticancer drugs are also exploited to improve tumor vaccination protocols. An important animal model used in these studies is FVB/ N-MMTV-neu transgenic mice developing mammary cancer due to overexpression of neu, the normal rat homologue of HER2/erbB2 in the mammary gland [7]. These mice are immunotolerant to neu [8,9], but can be vaccinated with neu-directed vaccines to prevent tumor formation in combination with appropriate adjuvants such as GM-CSF, IL-12, and cyclophosphamide [10,11]. The IFN-induced transcription factor Stat1 has been described as important mediator of the antitumor response [12]. As a key regulator of innate as well as adaptive immunity, Stat1 is involved in immune surveillance [13] but has also been postulated to act as a tumor suppressor by tumor epithelium intrinsic mechanisms. As has been shown recently, Stat1-deficient mice spontaneously develop mammary tumors [14,15]. In MMTV-neu mammary tumor mice, deletion of Stat1 in the tumor epithelium as well as in the tumor stroma was shown to contribute to accelerated tumorigenesis [16,17]. The aim of the present study was to investigate this issue further in MMTV-neu mice as an animal model for erbB2-positive breast cancer, treated in vivo with two different types of drugs, the dual tyrosine kinase inhibitor lapatini which targets HER2/erbB2/neu and EGFR/erbB1, and the genotoxic anthracycline drug doxorubicin.Here, we show that in MMTV-neu mice the in vivo efficacy of lapatinib and/or doxorubicin treatment is dependent on CD8 . Both Stat1-deficient and -proficient mice developed mammary tumors with no significant differences in expression levels for erbB2 and in tumor histology ( Fig. 1A and Supporting Information Fig. 1).As described previously for other mouse models of erbB2-positive breast cancer [16,17], Stat1 deficiency resulted in a slight acceleration of the development of palpable tumors. Furthermore, we observed an increase in tumor multiplicity, with lowered levels of caspase 3 cleavage in the tumor and a slight increase of the fraction of proliferating cells in the tumor (Supporting Information Fig. 1). To assess the role of Stat1 in the response to chemotherapy, mice were treated with the erbB1/erbB2 targeting drug lapatinib and/or the genotoxic agent doxorubicin as soon as tumors were palpable. The response to therapy was monitored for 6 weeks. Treatment of Stat1-proficient mice either with lapatinib alone, doxorubicin or drug combination resu...

Lactogenic hormone-dependent expression of the rat beta-casein gene in mammary epithelial cells is controlled via a complex regulatory region in the promoter. The sequence between -176 and -82 is the minimal region to confer the response to glucocorticoid hormone and prolactin on a heterologous promoter. The response is further enhanced by the region between -282 and -176. DNase I footprinting experiments and electromobility shift assays revealed the presence of four binding sites for CCAAT/enhancer-binding protein (C/EBP) isoforms in the hormone response region between -220 and -132. In nuclear extracts from mammary epithelial cells, the prevalent C/EBP isoform binding to these sites is beta (C/EBP-beta). C/EBP-delta is also present in mammary epithelial cells, whereas C/EBP-alpha is not detectable. The C/EBP sites are located in close proximity to the previously characterized binding sites for the prolactin-inducible mammary gland factor/signal transducer and activator of transcription-5, the nuclear factor YY1, and the glucocorticoid receptor. The importance of the two proximal C/EBP binding sites at the 5' border of the minimal region was tested by mutational analysis. Mutations of each site were found to inhibit strongly both the basal and the lactogenic hormone-induced transcription of a beta-casein gene promoter chloramphenicol acetyltransferase construct. The results implicate C/EBPs as important regulators of beta-casein gene expression in the mammary epithelium.

The mammary gland factor MGF has been described as a developmentally and environmentally regulated nuclear factor required for transcription of the milk protein gene beta-casein. In the current study the individual role of lactogenic hormones in the activation of MGF DNA binding and the functional relation of MGF to known transcription factors was investigated by electrophoretic mobility shift assays. DNA binding of MGF was rapidly induced by PRL in mammary epithelial cells. The activation was not inhibited by the protein synthesis inhibitor cycloheximide. The effect of PRL on MGF did not require costimulation of cells with the other lactogenic hormones, insulin, and glucocorticoids. Thus, MGF is the first example of a nuclear factor directly regulated by PRL. The MGF complexes formed upon initiation of lactation in the mammary gland and upon stimulation of mammary epithelial cells with PRL migrated at the same position in electrophoretic mobility shift assay, whereas the MGF complex found in mammary gland extracts of pregnant mice exhibited a faster mobility. In cell cultures, PRL-induced activation of MGF as well as up-regulation of beta-casein gene transcription was confined to confluent cultures of mammary epithelial cells and inhibited by long term incubation of cells with epidermal growth factor. MGF was found to be related to the nuclear factors that are activated by tyrosine phosphorylation when cells are stimulated with interferons or cytokines. This notion is supported by experimental evidence for phosphorylation of MGF on tyrosine and by the similar DNA recognition motifs of MGF and cytokine-activated factors.

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Regulatory regions have been located in the 5' flanking sequence of the mouse whey acidic protein gene which contribute to its tissue-and stage-specific expression in the mammary gland. They can be functionally separated into elements which mediate the action of lactogenic hormones prolactin and glucocorticoids and elements which control mammary cell-specific transcription in the absence of hormones. By mutational analysis, we have located a site in the whey acidic protein promoter between -120 and -100 which is important for hormone-independent promoter function, In stably transfected HCll mammary epithelial cells, the hormone-independent activity of the mutated promoter was reduced 40-fold, whereas the capability to respond to lactogenic hormones was retained. The site was specifically recognised by two nuclear factors contained in extracts of cultivated mammary epithelial cells or mammary glands. Electrophoretic mobility shift assay, DNase I footprinting and methylation interference experiments indicated a relation of both factors to the Ets family of DNA-binding proteins. One of these factors also recognised a functionally important site in the mammary cell-specific enhancer of the mouse mammary tumor virus long terminal repeat. The results suggest that factors related to the Ets family are important determinants in mammary cell-specific gene expression.Mammary epithelial cell-specific transcription of genes is under the control of complex cis-acting sequences, localised in the promoter region of the milk protein genes such as p-casein [l], P-lactoglobulin [2] and whey acidic protein (WAP) [3], or in the enhancer-like elements of the p-casein gene promoter [4] and in the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR) [5 -71. Numerous nuclear factors binding in vitro to these regions have been characterised 15, [8][9][10][11][12][13][14][15][16]. For some of these factors, the importance of their binding sites in the mediation of hormone-dependent [ 10, 12 -141 and independent mammary cell-specific transcription has been demonstrated [5].Functional analysis of the WAP gene promoter revealed that both lactogenic hormone-dependent and independent regulatory mechanisms contribute to its efficient transcription in mammary epithelial cells [3]. In detailed analyses of the regulatory elements involved [3, 51, the major hormoneindependent regulatory region was mapped to the sequence between positions -195 and +28 of the start of transcription of this gene. Part of this region has previously been shown to be highly conserved in the promoter region of four wheyCorrespondence to W, Doppler, Institut fur Medizinische Chemie und Biochemie, Fritz-Pregl-StraRe 3, A-6020 Innsbruck, AustriaFax: +43 512 517 2872. Abbreviations. CAT, chloramphenicol acetyltransferase; EGF, epidermal growth factor; EMSA, electrophoretic mobility shift assay ; MAF, mammary cell-activating factor; MMTV-LTR, mouse mammary tumor virus long terminal repeat ; NFAT, interleukin-2 enhancer; tk, thymidine kinase gene promoter of the he...